Functional role of cyclic nucleotide-gated channels in rat medial vestibular nucleus neurons

Abstract

Although cyclic nucleotide-gated (CNG) channels are expressed in numerous brain areas, little information is available on their functions in CNS neurons. The aim of the present study was to define the distribution of CNG channels in the rat medial vestibular nucleus (MVN) and their possible involvement in regulating MVN neuron (MVNn) excitability. The majority of MVNn expressed both CNG1 and CNG2 A subunits. In whole-cell current-clamp experiments carried out on brainstem slices containing the MVNn, the membrane-permeant analogues of cyclic nucleotides, 8-Br-cGMP and 8-Br-cAMP (1 mM), induced membrane depolarizations (8.9 +/- 0.8 and 9.2 +/- 1.0 mV, respectively) that were protein kinase independent. The cGMP-induced depolarization was associated with a significant decrease in the membrane input resistance. The effects of cGMP on membrane potential were almost completely abolished by the CNG channel blockers, Cd(2+) and L-cis-diltiazem, but they were unaffected by blockade of hyperpolarization-activated cyclic nucleotide-gated channels. In voltage-clamp experiments, 8-Br-cGMP induced non-inactivating inward currents (-22.2 +/- 3.9 pA) with an estimated reversal potential near 0 mV, which were markedly inhibited by reduction of extracellular Na(+) and Ca(2+) concentrations. Membrane depolarization induced by CNG channel activation increased the firing rate of MVNn without changing the action potential shape. Collectively, these findings provide novel evidence that CNG channels affect membrane potential and excitability of MVNn. Such action should have a significant impact on the function of these neurons in sensory-motor integration processes. More generally, it might represent a broad mechanism for regulating the excitability of different CNS neurons.

Figure 1. Expression patterns of CNG1 and CNG2 in coronal sections of MVN

A, CNG1 immunolabelling (green) of MVN neurons identified on the basis of immunoreactivity for NeuN (red). The right panel shows co-localization of the two markers in the majority of the MVNn. B, astrocytes (GFAP⁺ cells, red) do not express CNG1. C and D, staining for CNG2 (green) is observed in both neurons and astrocytes of the MVN. In the right panels of B and D, staining of cell nuclei with DAPI (blue) is also shown.

Figure 2. Effects of cyclic nucleotides on membrane potential of MVNn

A, whole-cell current-clamp recordings showing representative examples of membrane depolarization induced by 1 mm 8-Br-cGMP (top) and 1 mm 8-Br-cAMP (bottom). B, mean values of effects induced by 8-Br-cGMP and 8-Br-cAMP alone and in the presence of inhibitors of PKG (KT5823, 1 μm) and PKA (H89, 1 μm). C, voltage traces (top) and I–V curves (bottom) from a representative MVNn subjected to 600 ms hyperpolarizing current steps (from −40 pA to −10 pA in 10 pA increments) in control and during 8-Br-cGMP application. The input resistance of this cell, determined from the slope of linear regression analysis (continuous line), was 391 MΩ in control and 306 MΩ during 8-Br-cGMP application. D, plots of input resistance values in all cells tested (n = 7) in control conditions and during 8-Br-cGMP application. E, input resistance decrease induced by 8-Br-cGMP expressed as percentage of control values. Error bars show s.e.m. values *P < 0.05.

Figure 3. Cyclic nucleotide-gated currents in MVNn

A, representative inward current elicited by 1 mm 8-Br-cGMP in a MVNn held at −60 mV. B, mean amplitude of currents induced by 8-Br-cGMP and 8-Br-cAMP at −60 mV. C, I–V relationships built plotting data collected from slices perfused with 8-Br-cGMP in aCSF (^) or choline-aCSF (•). Net currents activated by cGMP at the various voltages were obtained by subtracting the currents recorded in control conditions to those recorded in the presence of the 8-Br-cGMP. Voltage steps (from −80 mV to −30 mV in 10 mV increments) were applied from holding potential of −60 mV. Data of cGMP-activated currents recorded in aCSF were fitted by linear regression line (R² = 0.99, P < 0.001). The estimated reversal potential (3.0 mV) was determined by extrapolation. D, representative trace showing inhibition of cGMP-activated current following replacement of aCSF with choline-aCSF. Error bars show s.e.m. values

Figure 4. Effects of CNG and HCN channel blockers on 8-Br-cGMP-induced depolarization

A, application of 3 mm Cd²⁺ reverted the effects of 8-Br-cGMP on V_m. B, inhibitory effects of Cd²⁺ on 8-Br-cGMP-induced membrane depolarizations in the five studied MVNn. C, representative trace showing the lack of 8-Br-cGMP effect in the presence of the CNG channel blocker LCD. D, mean values of membrane depolarizations induced by 8-Br-cGMP alone (n = 13) and in the presence of: the selective CNG channel blocker LCD (100 μm; n = 6); choline-aCSF (n = 5); and the selective HCN channel blocker ZD7288 (25 μm; n = 5). Error bars show s.e.m. values *P < 0.05; **P < 0.001.

Figure 5. Effects of 8-Br-cGMP on MVNn firing rate

A, in perforated-patch recordings 1 mm 8-Br-cGMP induced membrane depolarization associated with increase in the discharge frequency (middle trace). The 8-Br-cGMP effects were completely reversed by constant injection of hyperpolarizing currents, returning V_m to the predrug level value (right). Spikes were truncated at −30 mV for clarity. B, mean values of the firing rate in control conditions and during 8-Br-cGMP application with and without constant current injection in order to return the membrane potential to control value (n = 5). C, traces taken from the three conditions shown in A are superimposed according to their firing threshold to compare the shape of action potentials. The rate of the membrane depolarization in the interspike intervals was defined as the slope of the straight line drawn between the peak of AHP and the threshold for action potential. Values of interspike depolarization slopes are plotted in D and their changes expressed as a percentage of controls in E. Error bars show s.e.m. values *P < 0.05.